Use of MIR-92A or MIR-145 in the treatment of Angelman syndrome

ABSTRACT

Expression of micoRNAs that negatively regulate the activity of the SNHG14 gene for can be used in treatment of Angelman Syndrome. Such microRNAs include, for example, MIR-92a and/or MIR-145, as well as analogues and variants thereof, for use in treatment of Angelman Syndrome. Expression vectors such as, for example, AAV vectors may be used to transduce cells for introduction of MIR-92a and/or MIR-145 into target tissues for treatment of Angelman Syndrome.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit and priority to U.S. ProvisionalApplication No. 62/684,774, filed Jun. 14, 2018, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to expression of micoRNAs thatnegatively regulate the activity of the SNHG14 gene for use in treatmentof Angelman Syndrome. Such microRNAs include, for example, MIR-92aand/or MIR-145, as well as analogues and variants thereof, for use intreatment of Angelman Syndrome. More specifically, the presentdisclosure relates to the use of expression vectors such as, forexample, AAV vectors that may be used to transduce cells forintroduction of MIR-92a and/or MIR-145 into the target tissues fortreatment of Angelman Syndrome.

BACKGROUND

“Angelman syndrome” (AS) is a neurodevelopmental disorder characterizedby severe developmental delay or intellectual disability, severe speechimpairment, gait ataxia and/or tremulousness of the limbs, seizures,microcephaly and a unique behavior with an inappropriate happy demeanorthat includes frequent laughing, affinity for water, smiling, andexcitability. Microcephaly and seizures are also common. Developmentaldelays are first noted at around age six months; however, the uniqueclinical features of Angelman syndrome do not become manifest untilafter age one year, and it can take several years before the correctclinical diagnosis is obvious.

Many of the characteristic features of Angelman syndrome result from theloss of function of a gene called UBE3A which encodes for ubiquitinprotein ligase E3A (UBE3A) gene. (Kishino, T. et al. Nat Genet (1997)12:385-395). People normally inherit one copy of the UBE3A gene fromeach parent. Both copies of this gene are turned on (active) in many ofthe body's tissues, however, in certain areas of the brain, only thecopy inherited from a person's mother (the maternal copy) is active.This parent-specific gene activation is caused by a phenomenon calledgenomic imprinting. UBE3A is maternally imprinted in the brain, suchthat it is expressed nearly exclusively from the maternal chromosomewhile the paternal chromosome is epigenetically silenced. (Albrecht, U.et al., Nat Genet (1997) 17:75-78). If the maternal copy of the UBE3Agene is lost because of a chromosomal change or a gene mutation, aperson will have no active copies of the gene in some parts of thebrain.

Several different genetic mechanisms can inactivate or delete thematernal copy of the UBE3A gene. Most cases of Angelman syndrome (about70 percent) occur when a segment of the maternal chromosome 15containing this gene is deleted. In other cases (about 11 percent),Angelman syndrome is caused by a mutation in the maternal copy of theUBE3A gene.

Small nucleolar RNA host gene 14 (SNHG14), alternatively known asUBE3A-ATS, extends antisense into the UBE3A gene and thus plays apotential role in suppression of paternal UBE3A expression andimprinting. Much remains to be understood regarding how insufficiency ofthe protein product of UBE3A results in the observed neurodevelopmentaldeficits observed in Angelman Syndrome. Accordingly, there remains aneed for improved and/or additional therapies for treating subjectsdiagnosed with Angelman Syndrome.

SUMMARY

A method of treating Angelman Syndrome (AS) in a patient in need thereofis provided which includes delivering to the patient an effective amountof a composition that negatively regulates the activity of the SNHG14gene. Such methods of treatment include delivery to a patient aneffective amount of a composition that increases the level ofmicroRNA-145 and/or microRNA-92a molecules in the central nervous systemof the patient. The method disclosed herein is designed to negativelyregulate the activity of the SNHG14 gene.

A method of treating Angelman Syndrome in a patient in need thereof isprovided which includes delivering to the patient an effective amount ofa composition that increases the level of microRNA-145 and/ormicroRNA-92a molecules in cells of the central nervous system of thepatient. A method of treating Angelman Syndrome in a patient in needthereof is provided which includes administering a vector encodingmicroRNA-145, pri-miRNA145 or pre-miRNA145 to the patient. A method oftreating Angelman Syndrome in a patient in need thereof is providedwhich includes administering a vector encoding microRNA-92a, pri-miR92aor pre-miRNA92a. In embodiments, increased levels of microRNA-145 ormicroRNA-92a cause improvement in one or more symptoms of the AngelmanSyndrome.

In embodiments, a vector encoding microRNA-145, pri-miR145 orpre-miR145, causes increased levels of microRNA-145 in a patient withAngelman Syndrome and is associated with reduced symptoms of thedisorder. In embodiments, a vector encoding microRNA-92a, pri-miR92a orpre-miR92a causes increased levels of microRNA-92a in a patient withAngelman Syndrome and is associated with reduced symptoms of thedisorder.

In certain aspects, a vector including nucleic acids encodingmicroRNA-145, pri-miR145 or pre-miR145, includes a promoter operativelylinked to the nucleic acid encoding microRNA-145, pri-miR145 orpre-miR145. In embodiments, the vector includes a woodchuckpost-transcriptional regulatory element (WPRE). In embodiments, thevector includes a bovine growth hormone polyadenylation sequence(BGHpA). In embodiments, the vector includes a fluorescence reportercassette. In embodiments, the vector is an adeno-associated virus. Inembodiments, the vector is a lentivirus. In embodiments, a vectorincluding nucleic acid encoding microRNA-92a, pri-miR92a or pre-miR92a,includes a promoter operatively linked to the nucleic acid encodingmicroRNA-92a, pri-miR92a or pre-miR92a. In embodiments, the nucleic acidencoding microRNA-92a is microRNA-92a-3p. In embodiments, the vectorincludes a woodchuck post-transcriptional regulatory element (WPRE). Inembodiments, the vector includes a bovine growth hormone polyadenylationsequence (BGHpA). In embodiments, the vector includes a fluorescencereporter cassette. In embodiments, the vector is an adeno-associatedvirus. In embodiments, the vector is a lentivirus. In embodiments, thevector is an AAV vector.

In embodiments, the vector is delivered to a target location in thepatient's central nervous system. In embodiments, the target location isthe patient's brain. In embodiments, the route of administration of thevector is oral, buccal, sublingual, rectal, topical, intranasal, vaginalor parenteral. In embodiments, the vector is administered directly tothe target location.

In embodiments, ultrasound is applied to a target location in thepatient's brain to enhance permeability of the patient's blood brainbarrier at a target location, wherein microRNA-145 or microRNA-92a isdelivered to the target location.

DETAILED DESCRIPTION

Described herein are methods and compositions for treating AngelmanSyndrome which include administering compositions that negativelyregulate the activity of the SNGH14 gene. Such compositions include, forexample, microRNA-145, pri-miR145 or pre-miR145, to a patient havingAngelman Syndrome. Also described herein are methods and compositionsfor treating Angelman Syndrome which include administering microRNA-92a,pri-miR92a or pre-miR92a, to a patient having Angelman Syndrome.

In embodiments, vectors encoding microRNA-145, pri-miR145 or pre-miR145are provided. In embodiments, vectors encoding microRNA-145, pri-miR145or pre-miR145 are administered to a patient having Angelman Syndromewherein the patient exhibits improvement in one or more symptoms of thedisorder. In embodiments, vectors encoding microRNA-92a, pri-miR92a orpre-miR92a are provided. In embodiments, vectors encoding microRNA-92a,pri-miR92a or pre-miR92a are administered to a patient having AngelmanSyndrome wherein the patient exhibits improvement in one or moresymptoms of the disorder.

MicroRNA-145, pri-miR145, pre-miR145, microRNA-92a, pri-miR92a, and/orpre-miR92a, are collectively referred to herein as microRNA ormicroRNAs. Administration to a patient of microRNA-145, pri-miR145,pre-miR145, microRNA-92a, pri-miR92a, and/or pre-miR92a, is collectivelyreferred to herein as microRNA treatment. MicroRNA treatment increasesthe level of respective active microRNA molecules in a cell. Theincrease can come about by directly providing the microRNA to a cell, ormay come about by indirectly providing microRNA to cell, such as througha vector. The microRNA may include a RNA or DNA molecule that alsoincludes additional sequences. Increases in the level of respectiveactive microRNA molecules in cells of the CNS, for example, the brain ofa patient are associated with an improvement in one or more symptoms ofAngelman Syndrome.

One or more pri-miRNA(s) can be used in the compositions and methodsdescribed herein. Any suitable form of a pri-mRNA can be used. Thepri-mRNA(s) can be processed intracellularly and act to gain functionfor the miRNA, e.g., converted into pre-mRNA(s) and then the matureform. Alternatively, the miRNA may initially be a miRNA precursor. Inembodiments, the compositions and methods include pre-miRNA, which issubject to cleavage by an RNAse III type double stranded endonucleasecalled Dicer, resulting in an imperfect miRNA:miRNA* duplex that isabout 20-25 nucleotides in size. This duplex contains the mature miRNAstrand and its opposite complementary miRNA* strand. One or morepre-miRNA(s) can be used in the compositions and methods describedherein. The pre-miRNA may act to gain function for the miRNA. Anysuitable form of a pre-miRNA can be used. It is also contemplated thatthe miRNA of the compositions and methods described herein may be maturemiRNA.

The microRNAs can be delivered to cells in non-expression vector orexpression vector modalities. Expression vector and vector are usedinterchangeably herein. In embodiments, microRNA may be isolated orpurified prior to use in a subsequent step. MicroRNAs may be isolated orpurified prior to introduction into a cell. “Introduction” into a cellincludes known methods of transfection, transduction, infection andother methods for introducing an expression vector or a heterologousnucleic acid into a cell. A template nucleic acid or amplificationprimer may be isolated or purified prior to it being transcribed oramplified. Isolation or purification can be performed by a number ofmethods known to those of skill in the art with respect to nucleicacids. The delivery of the microRNA may occur through several forms,such as through encapsulation of a chemically modified or through anunmodified RNA moiety within a viral or non-viral delivery vessel.Non-expression vector delivery modalities include nanoparticles,microparticles, liposomes, polymers, microspheres, etc., which may betargeted to brain cells. The microRNA can also be delivered as a plasmidor mini-vector based expression system where it can then be expressedand processed by the RNAi machinery in cells to form a mature microRNA.

Nucleic acid constructs for miRNA expression may be producedrecombinantly. Such expression vectors are provided herein. Expressionvectors are a carrier nucleic acid into which a nucleic acid sequencecan be inserted for introduction into a cell where it can be replicated.Expression vectors include plasmids, cosmids, recombinant viruses, suchas adeno-associated virus (AAV), adenoviruses, retroviruses, poxviruses,and other known viruses in the art (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). A person ofordinary skill in the art is well equipped to construct expressionvectors through standard recombinant techniques. In embodiments, anexpression vector having a microRNA is delivered to cells of a patient.The nucleic acid molecules are delivered to the cells of a patient in aform in which they can be taken up and are advantageously expressed sothat therapeutically effective levels can be achieved.

Nucleic acid molecules for use in the vectors disclosed herein includethose encoding for mammalian microRNA-145, pri-miR145, pre-miR145,microRNA-92a, pri-miR92a, or pre-miR92a. Such nucleic acids are wellknown in the art and publically available. In an embodiment of theinvention, human microRNA-145, pri-miR145, pre-miR145, microRNA-92a,pri-miR92a, or pre-miR92a sequences are used in the vectors. The maturesequence for human microRNA-92a, also referred to as microRNA-92a-3p is:

(SEQ ID NO: 1) 5′-UAUUGCACUUGUCCCGGCCUGU-3′

The mature sequence for human microRNA-145, also referred to asmicroRNA-145-5p is:

(SEQ ID NO: 2) 5′GUCCAGUUUUCCCAGGAAUCCCU-3′

In addition to wild type microRNA-145 and microRNA-92a encoding nucleicacids, variants of said nucleic acids may also be used in the methods ofthe invention. Such variants may, for example, affect the localizationof the microRNA within the cell. Such variants may result in a reductionin the nuclear localization of the microRNA with an increase incytoplasmic localization. In an aspect of the invention, microRNA-145and microRNA-92a mimics may also be used to treat Angelman Syndrome.Such mimics are commercially available (See, Sigma Aldrich, forexample).

Any suitable expression vector known to those skilled in the art may beutilized to deliver microRNA(s) herein to a target location in cells ofthe central nervous system. Upon such delivery, cells in the targetlocations are transfected with microRNA(s), thereby increasing levels ofthose microRNA(s) in the brain of the patient. Transducing viral (e.g.,retroviral, adenoviral, lentiviral and adeno-associated viral) vectorscan be used for somatic cell gene therapy, especially because of theirhigh efficiency of infection and stable integration and expression.

In embodiments, the expression vector may be a stable integrating vectoror a stable nonintegrating vector. Examples of suitable vectors arelentiviruses and adeno-associated viruses (AAV). Lentiviruses are asubclass of retroviruses. Lentiviruses can integrate into the genome ofnon-dividing cells such as neurons. Lentiviruses are characterized byhigh-efficiency infection, long-term stable expression of transgenes andlow immunogenicity. In embodiments, lentiviral vectors may be utilizedto deliver microRNA(s) to the brain.

AAV is a defective parvovirus known to infect many cell types and isnonpathogenic to humans. AAV can infect both dividing and non-dividingcells. In embodiments, AAV vectors may be utilized herein to delivermicroRNA(s) to the brain. Any of the known adeno-associated viruses(AAV) may be utilized herein, e.g., AAV1, AAV2, AAV4, AAV5, AAV8, AAV9and AAVRec3 may be utilized in connection with neurons. AAV vectors foruse in the methods disclosed herein include those described in US SerialNo. U.S. Provisional Patent Application No. 62/550,458 which isincorporated by reference herein in its entirety. Additional suitableAAV serotypes have been developed through pseudotyping, i.e., mixing thecapsid and genome from different viral serotypes. Accordingly, e.g.,AAV2/7 indicates a virus containing the genome of serotype 2 packaged inthe capsid from serotype 7. Other examples are AAV2/5, AAV2/8, AAV2/9,etc. Hybrid AAV capsid serotypes rec1, rec2, rec3 and rec4 weregenerated by shuffling the fragments of capsid sequences that matched inall three non-human primate AAV serotypes cy5, rh20 and rh39, with AAV8.See, Charbel et al., PLoS One. 2013 Apr. 9; 8(4):e60361. The termsrec3AAV and AAVRec3 may be used interchangeably herein.Self-complementary adeno-associated virus (scAAV) may also be utilizedas vectors. Whereas AAV packages a single strand of DNA and requires theprocess of second-strand synthesis, scAAV packages both strands whichanneal together to form double stranded DNA. By skipping second strandsynthesis scAAV allows for rapid expression in the cell.

Suitable vectors may be constructed by those having ordinary skill inthe art using known techniques. Suitable vectors can be chosen orconstructed, containing, in addition to microRNA(s), appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, marker genes and other sequencesas appropriate. Those skilled in the art are familiar with appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, marker genes and other suitablesequences.

Expression vectors herein include appropriate sequences operably linkedto the coding sequence or ORF to promote its expression in a targetedhost cell. “Operably linked” sequences include both expression controlsequences such as promoters that are contiguous with the codingsequences and expression control sequences that act in trans or distallyto control the expression of the desired product.

Typically, the vector includes a promoter to facilitate expression ofthe microRNA(s) within a target cell. The promoter may be selected froma number of constitutive or inducible promoters that can driveexpression of the selected transgene in the brain. Examples ofconstitutive promoters include CMV immediate early enhancer/chickenbeta-actin (CBA) promoter-exon 1-intron 1 element, RSV LTRpromoter/enhancer, the SV40 promoter, the CMV promoter, dihydrofolatereductase (DHFR) promoter, and the phosphoglycerol kinase (PGK)promoter.

Specificity can be achieved by regional and cell-type specificexpression of the receptor exclusively, e.g., using a tissue or regionspecific promoter. Virus gene promoter elements may help dictate thetype of cells that express microRNA(s). Some promoters are nonspecific(e.g., CAG, a synthetic promoter), while others are neuronal-specific.The CAG promoter is a strong synthetic promoter that can be used todrive high levels of expression. The CAG promoter consists of 1) acytomegalovirus (CMV) early enhancer element, 2) the promoter, the firstexon and the first intron of the chicken beta-actin gene, and 3) thesplice acceptor of the rabbit beta-globin gene. In embodiments thepromoter is the CAG promoter. Neuronal specific promoters include (e.g.,synapsin; hSyn), or preferential to specific neuron types, e.g.,dynorphin, encephalin, GFAP (Glial fibrillary acidic protein) which ispreferential to astrocytes, or CaMKIIa, which is preferential tocortical glutamatergic cells but can also target subcortical GABAergiccells. In embodiments, the promoter is the CamkIIa (alpha CaM kinase IIgene) promoter, which may drive expression in the forebrain. Otherneuronal cell type-specific promoters include the NSE promoter, tyrosinehydroxylase promoter, myelin basic protein promoter, glial fibrillaryacidic protein promoter, and neurofilaments gene (heavy, medium, light)promoters.

Expression control sequences may also include appropriate transcriptioninitiation, termination, and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., Kozak consensus sequence); sequences thatenhance nucleic acid or protein stability; and when desired, sequencesthat enhance product processing and/or secretion. Many varied expressioncontrol sequences, including native and non-native, constitutive,inducible and/or tissue-specific, are known in the art and may beutilized herein depending upon the type of expression desired.

In addition to promoters, expression control sequences for eukaryoticcells typically include an enhancer, such as one derived from animmunoglobulin gene, SV40, CMV, etc., and a polyadenylation sequencewhich may include splice donor and acceptor sites. The polyadenylationsequence generally is inserted 3′ to the coding sequence and 5′ to the3′ ITR sequence. Illustrative examples of polyA signals that can be usedin a vector herein include polyA sequence (e.g., AATAAA, ATTAAA, orAGTAAA), a bovine growth hormone polyA sequence (BGHpA), a rabbitbeta-globin polyA sequence (rBgpA), or another suitable heterologous orendogenous polyA sequence known in the art.

Regulatory sequences useful herein may also contain an intron, such asone located between the promoter/enhancer sequence and the codingsequence. One useful intron sequence is derived from SV40, and isreferred to as the SV40 T intron sequence. Another includes thewoodchuck hepatitis virus post-transcriptional element (WPRE). WPRE is aDNA sequence that, when transcribed, creates a tertiary structure thatenhances expression.

Vectors herein may contain reporter genes, e.g., those which encodefluorophores. A fluorophore is a fluorescent compound that can re-emitlight upon excitation, usually at specific frequencies. They can be usedas a tag or marker which can be attached to, e.g., a protein to allowthe protein to be located. Many suitable fluorophores are known in theart. They may be categorized by the color they emit, e.g., blue, cyan,green, yellow, orange, red and others. For example, mCherry, mRasberry,mTomato and mRuby are red fluorophore proteins; citrine, venus, and EYFPare yellow fluorophore proteins. Green fluorescent protein (GFP) is acommonly used fluorophore.

The microRNAs described herein, whether delivered by expression vectoror by non-expression vector modalities, are used to treat AngelmanSyndrome. Symptoms of Angelman Syndrome may include, but are not limitedto intellectual disability, lack of speech, seizures, and acharacteristic behavioral profile. The behavioral features of AngelmanSyndrome include a happy demeanor, easily provoked laughter, shortattention span, hypermotoric behavior, mouthing of objects, sleepdisturbance, and an affinity for water. Methods of treatment herein caninclude providing improvement in one or more of the foregoing symptoms.

In certain aspects, a patient suspected of carrying a genetic defectresulting in Angelman Syndrome may be tested prior to treatment todetect and confirm the presence of such a defect. In one example, thepatient may be tested to detect defects in the UBE3A gene. Moleculargenetic testing (methylation testing and UBE3A sequence analysis) iscapable of identifying alterations in approximately 90% of individuals.

Once a determination has been made of the location of a suspectedlocation of abnormal activity associated with Angelman Syndrome in apatient, targeted treatment in accordance with the present disclosurecan be implemented. Methods of determining the location of abnormalactivity in the brain, or additionally affected neuronal tissue, arewell-known in the art. In embodiments, areas determined to be the siteof origin of the abnormal activity can be targeted.

In some embodiments, the vectors disclosed herein are administereddirectly to the central nervous system, e.g., the brain or the spinalcord. Any method known in the art to administer vectors directly to thecentral nervous system can be used. The vectors may be introduced intothe spinal cord, brainstem (medulla oblongata, pons), midbrain(hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra,pineal gland), cerebellum, telencephalon (corpus striatum, cerebrumincluding the occipital, temporal, parietal and frontal lobes, cortex,basal ganglia, hippocampus and amygdala), limbic system, neocortex,corpus striatum, cerebrum, and inferior colliculus. The vectors may bedelivered into the cerebrospinal fluid by, for example, lumbar puncture.In an addition, when administration is performed intravenously,ultrasound may be applied to a target location in the patient's brain toenhance permeability of the patient's blood brain barrier at the targetlocation for uptake of the vectors. The application of ultrasound forenhancing the permeability of the patient's blood brain barrier isdisclosed in Ser. No. 62/471,635, the content of which is incorporatedherein in its entirety.

Methods for administering materials directly to target locations such asthe brain, are well-known. For example, a hole, e.g., Burr hole, can bedrilled into the skull and an appropriately sized needle may be used todeliver a vector or non-vector vehicle to a target location. Inembodiments, a portion of the skull may be removed to expose the duramatter (craniotomy) at or near a target location and a vector ornon-vector vehicle can be administered directly to the target location.In embodiments, a vector or non-vector vehicle is injectedintracranially using stereotaxic coordinates, a micropipette and anautomated pump for precise delivery of the vector or non-vector vehicleto the desired area with minimal damage to the surrounding tissue.

In certain aspects, a micropump may be utilized to deliverpharmaceutical compositions containing a vector or non-vector vehiclecontaining the microRNA(s) to target areas in the brain. Thecompositions can be delivered immediately or over an extended period oftime, e.g., over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more minutes. Aftervector delivery to a target location in the brain a sufficient amount oftime may be allowed to pass to allow expression of the microRNA(s) atthe target location.

In certain aspects, vectors or nonvector delivery vehicles herein can beadministered systemically. Systemic delivery includes oral, buccal,sublingual, rectal, topical, intranasal, vaginal and parenteral modes ofadministration. Examples of parenteral modes of administration includeintravenous, intraperitoneal, intramuscular and subcutaneous modes ofadministration. In embodiments, vectors or nonvector delivery vehicleswill circulate until they contact the target location(s) in the CNS,including the brain, where they deliver the microRNA(s) or cause themicroRNA(s) to be expressed and act, e.g., to aid in network formationand/or modulate neuronal signaling networks.

The microRNA(s) is used in an amount effective against Angelman Syndromein patients. The dosage of the active ingredient depends upon the age,weight, and individual condition of the patient, the individualpharmacokinetic data, and the mode of administration. In the case of anindividual human having a bodyweight of about 70 kg the daily doseadministered of a microRNA can be from 0.01 mg/kg bodyweight to 100mg/kg bodyweight, e.g., from 0.1 mg/kg bodyweight to 50 mg/kgbodyweight, from 1 mg/kg to 20 mg/kg bodyweight administered as a singledose or as several doses.

In embodiments, treatment with ultrasound is used to enhance delivery ofthe microRNA(s) to target locations in the brain by disrupting the bloodbrain barrier. Use of focused ultrasound energy herein disrupts the BBBwithout adversely affecting the vector, non-vector delivery vehicle, themicroRNA(s), and/or brain tissue itself. Use of ultrasound energy hereincan increase the speed of delivery of vectors, non-vector deliveryvehicles, and/or the microRNA(s) to target locations in the brain,reduce side effects which may be associated with delivery of vectorsnon-vector delivery vehicles, and/or the microRNA(s) to target locationsin the brain, reduce dosage amounts while concentrating vectors,non-vector delivery vehicles, and/or the microRNA(s) at a targetlocation and can allow controlled release of the amount of vectors,non-vector delivery vehicles, and/or the microRNA(s) at a targetlocation. Methods for delivering ultrasound energy through the skull areknown in the art. See, e.g., U.S. Pat. No. 5,752,515 and US PublicationNo. 2009/0005711, both of which are hereby incorporated by reference intheir respective entireties. See also, Hynynen et al., NeuroImage 24(2005) 12-120.

In accordance with the present disclosure, microRNA treatment providesimprovement in one or more symptoms of Angelman Syndrome for more than 1hour after administration to the patient. In embodiments, microRNAtreatment provides improvement in one or more symptoms of the disorderfor more than 2 hours after administration to the patient. Inembodiments, microRNA treatment provides improvement in one or moresymptoms of the disorder for more than 3 hours after administration tothe patient. In embodiments, microRNA treatment provides improvement inone or more symptoms of the disorder for more than 4 hours afteradministration to the patient. In embodiments, microRNA treatmentprovides improvement in one or more symptoms of the disorder for morethan 6 hours after administration to the patient. In embodiments,microRNA treatment provides improvement in one or more symptoms of thedisorder for more than 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours afteradministration to the patient. In embodiments, improvement in at leastone symptom for 12 hours after administration to the patient is providedin accordance with the present disclosure. In embodiments, microRNAtreatment provides improvement of next day functioning of the patient.For example, the microRNA may provide improvement in one or moresymptoms of the disorder for more than about, e.g., 2 hours, 4 hours, 6hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20hours, 22 hours or 24 hours after administration and waking from a nightof sleep.

In embodiments, the methods described herein are effective to reduce,delay, or prevent one or more other clinical symptoms of AngelmanSyndrome. For example, the effect in a patient of microRNA treatment ina target location of the central nervous system, including the brain,can be compared to an untreated patient, or the condition of the patientprior to treatment. In embodiments, a symptom, pharmacologic, and/orphysiologic indicator is measured in a patient prior to treatment, andagain one or more times after treatment is initiated. In embodiments,the control is a reference level, or average determined based onmeasuring the symptom, pharmacologic, or physiologic indicator in one ormore patients that do not have the disease or condition to be treated(e.g., healthy patients). In embodiments, the amount of miR-145 and/ormiR-92a in brain tissue prior to treatment is compared to the amount ofmiR-145 and/or miR-92a in brain tissue after treatment. In embodiments,the effect of the treatment is compared to a conventional treatment thatis within the purview of those skilled in the art.

Effective treatment of Angelman Syndrome as disclosed herein may beestablished by showing reduction in the frequency or severity ofsymptoms (e.g., more than 10%, 20%, 30% 40% or 50%) after a period oftime compared with baseline. For example, after a baseline period of 1month, the patients having microRNA treatment may be randomly allocateda placebo as add-on therapy to standard therapies, during a double-blindperiod of 2 months. Primary outcome measurements may include thepercentage of responders on a microRNA and on placebo, defined as havingexperienced at least a 10% to 50% reduction of symptoms during thesecond month of the double-blind period compared with baseline.

In embodiments, pharmaceutical compositions containing vectors,non-vector delivery vehicles, and/or the microRNA(s) may be providedwith conventional release or modified release profiles. Pharmaceuticalcompositions may be prepared using a pharmaceutically acceptable“carrier” composed of materials that are considered safe and effective.The “carrier” includes all components present in the pharmaceuticalformulation other than the active substance or ingredients. The term“carrier” includes, but is not limited to, diluents, binders,lubricants, disintegrants, fillers, and coating compositions. Those withskill in the art are familiar with such pharmaceutical carriers andmethods of compounding pharmaceutical compositions using such carriers.

In embodiments, pharmaceutical compositions containing vectors,non-vector delivery vehicles, and/or the microRNA(s) are suitable forparenteral administration, including, e.g., intramuscular (i.m.),intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), orintrathecal (i.t.). Parenteral compositions must be sterile foradministration by injection, infusion or implantation into the body andmay be packaged in either single-dose or multi-dose containers. Inembodiments, liquid pharmaceutical compositions for parenteraladministration to a patient include an active substance, e.g., vectors,non-vector delivery vehicles, and/or the microRNA(s), in any of therespective amounts described above. In embodiments, the pharmaceuticalcompositions for parenteral administration are formulated as a totalvolume of about, e.g., 0.1 ml, 0.25 ml, 0.5 ml, 0.75 ml, 1 ml, 1.25 ml,1.5 ml, 1.75 ml, 2 ml, 2.25 ml, 2.5 ml, 2.75 ml, 3 ml, 3.25 ml, 3.5 ml,3.75 ml, 4 ml, 4.25 ml, 4.5 ml, 4.75 ml, 5 ml, 10 ml, 20 ml, 25 ml, 50ml, 100 ml, 200 ml, 250 ml, or 500 ml. In embodiments, the volume ofpharmaceutical compositions containing expression vectors are microliteramounts. For example, 0.1 microliters to 10 or more microliters can beinjected. For example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25,4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 8.25, 8.5,8.75, 9.0, 9.25, 9.5, 9.75, or 10 microliters. In embodiments, thecompositions are contained in a micropipette, a bag, a glass vial, aplastic vial, or a bottle.

In embodiments, pharmaceutical compositions for parenteraladministration include respective amounts described above. Inembodiments, pharmaceutical compositions for parenteral administrationinclude about 0.0001 mg to about 500 mg active substance, e.g., vectors,non-vector delivery vehicles, and/or the microRNA(s). In embodiments,pharmaceutical compositions for parenteral administration to a patientinclude an active substance, e.g., vectors, non-vector deliveryvehicles, and/or the microRNA(s), at a respective concentration of about0.001 mg/ml to about 500 mg/ml. In embodiments, the pharmaceuticalcomposition for parenteral administration includes an active substanceat a respective concentration of, e.g., about 0.005 mg/ml to about 50mg/ml, about 0.01 mg/ml to about 50 mg/ml, about 0.1 mg/ml to about 10mg/ml, about 0.05 mg/ml to about 25 mg/ml, about 0.05 mg/ml to about 10mg/ml, about 0.05 mg/ml to about 5 mg/ml, or about 0.05 mg/ml to about 1mg/ml. In embodiments, the pharmaceutical composition for parenteraladministration includes an active substance at a respectiveconcentration of, e.g., about 0.05 mg/ml to about 15 mg/ml, about 0.5mg/ml to about 10 mg/ml, about 0.25 mg/ml to about 5 mg/ml, about 0.5mg/ml to about 7 mg/ml, about 1 mg/ml to about 10 mg/ml, about 5 mg/mlto about 10 mg/ml, or about 5 mg/ml to about 15 mg/ml.

In embodiments, a pharmaceutical composition for parenteraladministration is provided wherein the pharmaceutical composition isstable for at least six months. In embodiments, the pharmaceuticalcompositions for parenteral administration exhibit no more than about 5%decrease in active substance for at least, e.g., 3 months or 6 months.In embodiments, the amount of vector or non-vector vehicle, degrades atno more than about, e.g., 2.5%, 1%, 0.5% or 0.1%. In embodiments, thedegradation is less than about, e.g., 5%, 2.5%, 1%, 0.5%, 0.25%, 0.1%,for at least six months.

In embodiments, pharmaceutical compositions for parenteraladministration are provided wherein the pharmaceutical compositionremains soluble. In embodiments, pharmaceutical compositions forparenteral administration are provided that are stable, soluble, localsite compatible and/or ready-to-use. In embodiments, the pharmaceuticalcompositions herein are ready-to-use for direct administration to apatient in need thereof.

The pharmaceutical compositions for parenteral administration providedherein may include one or more excipients, e.g., solvents, solubilityenhancers, suspending agents, buffering agents, isotonicity agents,stabilizers or antimicrobial preservatives. When used, the excipients ofthe parenteral compositions will not adversely affect the stability,bioavailability, safety, and/or efficacy of a vector, non-vectordelivery vehicle, and/or the microRNA(s), used in the composition. Thus,parenteral compositions are provided wherein there is no incompatibilitybetween any of the components of the dosage form.

In embodiments, parenteral compositions including vectors, non-vectordelivery vehicles, and/or the microRNA(s) include a stabilizing amountof at least one excipient. For example, excipients may be selected fromthe group consisting of buffering agents, solubilizing agents, tonicityagents, antioxidants, chelating agents, antimicrobial agents, andpreservative. One skilled in the art will appreciate that an excipientmay have more than one function and be classified in one or more definedgroup.

In embodiments, parenteral compositions include a vector, non-vectordelivery vehicle, and/or the microRNA(s) and an excipient wherein theexcipient is present at a weight percent (w/v) of less than about, e.g.,10%, 5%, 2.5%, 1%, or 0.5%. In embodiments, the excipient is present ata weight percent between about, e.g., 1.0% to 10%, 10% to 25%, 15% to35%, 0.5% to 5%, 0.001% to 1%, 0.01% to 1%, 0.1% to 1%, or 0.5% to 1%.In embodiments, the excipient is present at a weight percent betweenabout, e.g., 0.001% to 1%, 0.01% to 1%, 1.0% to 5%, 10% to 15%, or 1% to15%.

In embodiments, parenteral compositions may be administered as needed,e.g., once, twice, three, four, five, six or more times daily, orcontinuously depending on the patient's needs.

In embodiments, parenteral compositions of an active substance areprovided, wherein the pH of the composition is between about 4.0 toabout 8.0. In embodiments, the pH of the compositions is between, e.g.,about 5.0 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0.In embodiments, the pH of the compositions is between, e.g., about 6.5to about 7.5, about 7.0 to about 7.8, about 7.2 to about 7.8, or about7.3 to about 7.6. In embodiments, the pH of the aqueous solution is,e.g., about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.7,about 7.8, about 8.0, about 8.2, about 8.4, or about 8.6.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosure herein belongs.

The term “about” or “approximately” as used herein means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” can mean within 3 or more than 3 standarddeviations, per the practice in the art. Alternatively, “about” can meana range of up to 20%, up to 10%, up to 5%, and/or up to 1% of a givenvalue. Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value.

“Improvement” refers to the treatment of Angelman Syndrome.

“Improvement in next day functioning” or “wherein there is improvementin next day functioning” refers to improvement after waking from anovernight sleep period wherein the beneficial effect of administrationof microRNA therapy to a patient applies to at least one symptom of asyndrome or disorder herein and is discernable, either subjectively by apatient or objectively by an observer, for a period of time, e.g., 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, etc.after waking.

“Treating”, “treatment” or “treat” can refer to the following:alleviating or delaying the appearance of clinical symptoms of a diseaseor condition in a patient that may be afflicted with or predisposed tothe disease or condition, but does not yet experience or displayclinical or subclinical symptoms of the disease or condition. In certainembodiments, “treating”, “treat” or “treatment” may refer to preventingthe appearance of clinical symptoms of a disease or condition in apatient that may be afflicted with or predisposed to the disease orcondition, but does not yet experience or display clinical orsubclinical symptoms of the disease or condition. “Treating”, “treat” or“treatment” also refers to inhibiting the disease or condition, e.g.,arresting or reducing its development or at least one clinical orsubclinical symptom thereof “Treating”, “treat” or “treatment” furtherrefers to relieving the disease or condition, e.g., causing regressionof the disease or condition or at least one of its clinical orsubclinical symptoms. The benefit to a patient to be treated may bestatistically significant, mathematically significant, or at leastperceptible to the patient and/or the physician. Nonetheless,prophylactic (preventive) treatment and therapeutic (curative) treatmentare two separate embodiments of the disclosure herein.

“Pharmaceutically acceptable” refers to molecular entities andcompositions that are “generally regarded as safe”, e.g., that arephysiologically tolerable and do not typically produce an allergic orsimilar untoward reaction, such as gastric upset and the like, whenadministered to a human. In embodiments, this term refers to molecularentities and compositions approved by a regulatory agency of the federalor a state government, as the GRAS list under section 204(s) and 409 ofthe Federal Food, Drug and Cosmetic Act, that is subject to premarketreview and approval by the FDA or similar lists, the U.S. Pharmacopeiaor another generally recognized pharmacopeia for use in animals, andmore particularly in humans.

“Effective amount” or “therapeutically effective amount” can mean adosage sufficient to alleviate one or more symptoms of a syndrome,disorder, disease, or condition being treated, or to otherwise provide adesired pharmacological and/or physiologic effect. “Effective amount” or“therapeutically effective amount” may be used interchangeably herein.

“Co-administered with”, “administered in combination with”, “acombination of” or “administered along with” may be used interchangeablyand mean that two or more agents are administered in the course oftherapy. The agents may be administered together at the same time orseparately in spaced apart intervals. The agents may be administered ina single dosage form or in separate dosage forms.

“Patient in need thereof” may include individuals, e.g., mammals such ashumans, canines, felines, porcines, rodents, etc., that have beendiagnosed with Angelman Syndrome The methods may be provided to anyindividual including, e.g., wherein the patient is a neonate, infant, apediatric patient (6 months to 12 years), an adolescent patient (age12-18 years) or an adult (over 18 years). Patients include mammals. Suchpatients include those, for example, diagnosed with a genetic defect inthe Ube3a gene.

“Prodrug” refers to a pharmacological substance (drug) that isadministered to a patient in an inactive (or significantly less active)form. Once administered, the prodrug is metabolized in the body (invivo) into a compound having the desired pharmacological activity.

“Analog” and “Derivative” may be used interchangeably and refer to acompound that possesses the same core as the parent compound, but maydiffer from the parent compound in bond order, the absence or presenceof one or more atoms and/or groups of atoms, and combinations thereof.Enantiomers are examples of derivatives. The derivative can differ fromthe parent compound, for example, in one or more substituents present onthe core, which may include one or more atoms, functional groups, orsubstructures. In general, a derivative can be imagined to be formed, atleast theoretically, from the parent compound via chemical and/orphysical processes.

The term “pharmaceutically acceptable salt”, as used herein, refers toderivatives of the compounds defined herein, wherein the parent compoundis modified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,nontoxic base addition salts with inorganic bases. Suitable inorganicbases such as alkali and alkaline earth metal bases include metalliccations such as sodium, potassium, magnesium, calcium and the like. Thepharmaceutically acceptable salts can be synthesized from the parentcompound by conventional chemical methods.

It should be understood that the examples and embodiments providedherein are exemplary examples embodiments. Those skilled in the art willenvision various modifications of the examples and embodiments that areconsistent with the scope of the disclosure herein. Such modificationsare intended to be encompassed by the claims.

What is claimed is:
 1. A method for treating Angelman Syndrome in apatient in need thereof, comprising administering to the patient avector including a nucleic acid encoding microRNA-145, pri-miR145,pre-miR145, microRNA-92a, pri-miR-92a or pre-miRNA-92a, wherein saidnucleic acid is operably linked to a promoter, wherein one or moresymptoms of the Angelman Syndrome are improved.
 2. The method accordingto claim 1, wherein after the administration, expression ofmicroRNA-145, pri-miR145, pre-miR145, microRNA-145, pri-miR145,pre-miR145, microRNA-92a, pri-miR-92a or pre-miRNA-92a in the patient isassociated with reduced symptoms of the Angelman Syndrome.
 3. The methodaccording to claim 1, wherein the promoter is selected from the groupconsisting of CAG promoter, CMV promoter, human synapsin 1 gene promoter(hSyn), dynorphin promoter, encephalin promoter and CaMKII promoter. 4.The method according to claim 1, wherein the promoter is a CAG promoter.5. The method according to claim 1, wherein the vector includes awoodchuck post-transcriptional regulatory element (WPRE).
 6. The methodaccording to claim 1, wherein the vector includes a bovine growthhormone polyadenylation sequence (BGHpA).
 7. The method according toclaim 1, wherein the vector includes a fluorescence reporter cassette.8. The method according to claim 1, wherein the vector is anadeno-associated virus (AAV).
 9. The method according to claim 8,wherein the adeno-associated virus is AAV1, AAV2, AAV4, AAV5, AAV7,AAV8, AAV9 or AAVRec3.
 10. The method according to claim 1, wherein thevector is a lentivirus.
 11. The method according to claim 1, wherein thevector is delivered to a target location in the patient's brain.
 12. Themethod according to claim 1, wherein the vector is administered via aroute selected from the group consisting of oral, buccal, sublingual,rectal, topical, intranasal, vaginal and parenteral.
 13. The methodaccording to claim 1, wherein the vector is administered directly to thetarget location.
 14. The method according to claim 1, wherein the methodprovides improvement in at least one symptom selected from the groupconsisting of developmental delay, intellectual disability, speechimpairment, gait ataxia and/or tremulousness of the limbs, seizures,microcephaly, an inappropriate happy demeanor, frequent laughing,affinity for water, smiling, and excitability.
 15. The method accordingto claim 1, further comprising applying ultrasound to a target locationin the patient's brain to enhance permeability of the patient's bloodbrain barrier at the target location, wherein the vector is delivered tothe target location.
 16. A method of treating Angelman Syndrome in apatient in need thereof comprising administering to the patient aneffective amount of a pharmaceutical composition that increases thelevel of microRNA-145 and/or microRNA-92a molecules in the patient'sbrain, wherein the composition includes microRNA-145, pri-miR145,pre-miR145, microRNA-92a, pri-miR92a or pre-miR92a, or the compositionincludes a vector including nucleic acid encoding microRNA-145,pri-miR145, pre-miR145, microRNA-92a, pri-miR92a or pre-miR92a.
 17. Themethod according to claim 16, wherein after the administering,expression of microRNA-145, pri-miR145, pre-miR145, microRNA-92a,pri-miR92a or pre-miR92a in the patient is associated with reducedsymptoms of the Angelman Syndrome disorder, the symptoms being at leastone symptom selected from the group consisting of developmental delay,intellectual disability, speech impairment, gait ataxia and/ortremulousness of the limbs, seizures, microcephaly, an inappropriatehappy demeanor, frequent laughing, affinity for water, smiling, andexcitability.
 18. The method according to claim 16, wherein the nucleicacid encoding microRNA-145, pri-miR145, pre-miR145, microRNA-92a,pri-miR92a or pre-miR92a is operably linked to a promoter.
 19. Themethod according to claim 18, wherein the promoter is selected from thegroup consisting of CAG promoter, CMV promoter, human synapsin 1 genepromoter (hSyn), dynorphin promoter, encephalin promoter and CaMKIIpromoter.
 20. The method according to claim 16, wherein the vectorincludes a woodchuck post-transcriptional regulatory element (WPRE). 21.The method according to claim 16, wherein the vector includes afluorescence reporter cassette.
 22. The method according to claim 16,wherein the vector is an adeno-associated virus (AAV).
 23. The methodaccording to claim 22, wherein the adeno-associated virus is AAV1, AAV2,AAV4, AAV5, AAV7, AAV8, AAV9 or AAVRec3.
 24. The method according toclaim 16, wherein the vector is a lentivirus.
 25. The method accordingto claim 16, wherein the vector is delivered to a target location in thepatient's brain.
 26. The method according to claim 16, wherein thevector is administered via a route selected from the group consisting oforal, buccal, sublingual, rectal, topical, intranasal, vaginal andparenteral.
 27. The method according to claim 16, wherein the vector isadministered directly to the target location.
 28. The method of claim16, wherein the composition includes a vector that is a non-viralvector.
 29. The method of claim 28, wherein the non-viral vector is aliposome mediated delivery vector.